Abstract The opportunistic pathogen Clostridium difficile causes almost half a million infections and ~30,000 deaths in the United States annually, with an estimated economic cost of $6 billion. C. difficile infections (CDI) have symptoms ranging from mild diarrhea to fatal pseudomembrane colitis. CDI are facilitated through several secreted virulence factors, notably, toxins A and B (TcdA and TcdB, respectively), which inhibit host small GTPases. Currently, the typical treatment for CDI is a course of antibiotics, but ~20-25% of patients suffer recurrent infections after antibiotic regimens. Recently, ZINPLAVA (an anti-TcdB monoclonal antibody) has been approved by the FDA for reducing the recurrence of CDI, although the recurrence of infection remains at 15-17% with this new therapy. Thus, new treatment options are needed to combat CDI. In order to rationally design new proteins or molecules against CDI, the structures of its toxins need to be determined. The current challenge for structural characterization of TcdA and TcdB are their inherent flexibility, which has limited crystallographic studies to individual domains, or truncations of the proteins. In this project we propose to use cryo-electron microscopy (cryo-EM) to structurally characterize the full-length TcdB. One key part of TcdB required for toxicity is a potentially flexible region (a 97 amino acid segment called D97) near the end of the delivery domain. D97 is proposed to act as a hinge that shifts upon the pH change during the endocytosis of the toxin, although the mechanism is unclear as it remains to be structurally characterized. Based on our preliminary data, we have identified the designed ankyrin repeat protein (DARPin) DLD-4, a non-antibody anti- toxin protein that inhibits TcdB, showing promise as a novel treatment for CDI. We hypothesize that DARPin DLD-4 binds specific regions, including the essential D97 region in TcdB, to neutralize its toxicity through blocking its attachment to the receptors, and/or preventing its structural rearrangement to deliver the effector domain during endosomal acidification. In order to test this hypothesis we have developed three aims. In Aim 1, we will solve high-resolution structure of the holo-TcdB and reveal the structural mechanism of how DARPin DLD-4 neutralizes TcdB, which will guide the improvement of DLD-4 for better TcdB neutralization efficiency. In Aim 2, we will determine the structure of TcdB in complex with one of its colonic epithelial cellular receptors, the frizzled protein 2. We will then use a cell-binding assay to test how DLD-4 may block the cell attachment of TcdB. In Aim 3, we will visualize the structural rearrangement of holo-TcdB at pH=4.5, free or inserted into liposomes, to reveal the mechanism of pore formation, which delivers the effector domain. We will then use a cell-based assay to test the effect of DLD-4 in blocking the pH-induced pore formation by TcdB. Completion of the proposed aims will reveal the mechanistic details of the attachment and activation of the toxin during CDI as well as enabling researchers to rationally design molecules/proteins to treat symptoms caused by the secreted toxins.